Project Summary Latent HIV infection of resting CD4 T cells present a formidable challenge in the pursuit of treatments to purge HIV from the body. Latency is established and regulated in large part through the modifications of the nucleosomes that are bound around the viral promoter region. Drugs that reactivate latent HIV must influence these structures in order to allow HIV transcription and virus expression. However, for unknown reasons not all intact proviruses respond to latency reversing agents and thus present a particularly troublesome barrier to cure. A greater understanding of the regulation of HIV-1 chromatin, the installation of nucleosomes and their histone post translational modifications (PTM) will contribute greatly to the development of therapeutic control over HIV- 1 latency. This project seeks such an understanding and builds upon extensive preliminary studies revealing important new insights into these processes. For the first time we investigate the initial stages of HIV-1 chromatin installation, finding that it occurs either contemporaneously or soon after reverse transcription, and before integration into the cellular DNA. We find that repressive chromatin is installed in the absence of the viral protein Vpr being delivered in the virion. Virion Vpr dramatically increases the number of transcriptionally active proviruses in the first 4 days after infection of resting CD4 T cells. When Vpr is present, the nucleosomes that control HIV expression are modified in ways that facilitate HIV replication and block installation of repressive chromatin. For example, the alternate histone H2A.Z is installed only when virion Vpr is available, and H2A.Z is a central organizer of paused but responsive promoters. Without Vpr, the chromatin of latent proviruses takes on a more repressed structure, resulting in more proviruses that do not respond to latency reversing agents. Novel RNA-seq data demonstrate that virion Vpr reprograms gene expression pathways central to chromatin organization and transcriptional regulation. This project will describe both the initial steps in HIV chromatization and the long term processes that lead to reversible latency and irreversible repression. Aim 1 will systematically analyze early proviral chromatin in resting CD4 T cells and the influence of Vpr and Tat-directed transcription on the epigenetic landscape. Our central hypothesis is that Vpr directs installation of the transcriptional pre-initiation complex for basal pre-Tat transcription in resting T cells. Aim 2 will analyze Vpr-dependent pathways leading to these structures. RNA-seq data will be expanded and used to study novel Vpr targets of regulation, and known Vpr pathways will be investigated for their influence on early events. Aim 3 will examine long term infection and latency under the influence the structures and pathways gleaned from Aims 1 and 2. Our hypothesis is that Vpr protects the provirus from epigenetic repression and irreversible latency. The proposed studies will provide much needed information that will assist in the development of therapeutics that can purge HIV from the body or permanently repress virus replication without continual antiviral treatments.